The present invention relates to a novel nucleotide, nucleotide block and oligonucleotide, and a method for producing the nucleotide block and oligonucleotide. These compounds and production method thereof according to the present invention are useful in synthetic organic chemistry, biochemistry and pharmaceutical industries, for example, as intermediates for the production of oligonucleotides and a method for producing the oligonucleotides.
Hitherto, a solid phase preparation method has been employed for preparation of oligodeoxyribonucleotides and oligoribonucleotides. According to this method, DNA chain or RNA chain is sequentially extended on a solid phase carrier insoluble in various organic solvents. More specifically, in general, using a starting material of a nucleoside whose 3xe2x80x2-hydroxyl group is fixed on an insoluble carrier such as a porous glass, an oligonucleotide chain is extended from 3xe2x80x2 terminal to the direction of 5xe2x80x2 terminal by one base at a time, and according to this method, oligonucleotides of desired sequences can be synthesized (Koester et al, JP-B-62-50479 corresponding to PCT/WO85/00816 and U.S. Pat. No. 4,725,677, and Caruthers et al, JP-B-63-28439 corresponding to U.S. Pat. No. 4,415,732). The above solid phase synthesis method has the merit that the excessively used reagents or solvents can be readily removed at the time of extension of the chain, and each elementary reaction is allowed to proceed by using excess reagents as required, whereby oligodeoxyribonucleotides and oligoribonucleotides having the desired sequence can be produced.
Generally, xcex2-cyanophosphoramidite developed by Koester et al is used as a nucleotide reagent for extension of DNA chain or RNA chain, and a porous glass is used as a solid phase carrier. See H. Koester et al, xe2x80x9cTetrahedron Lett.xe2x80x9d, 52, 5843 (1983) and PCT/WO97/42202.
Furthermore, as the similar nucleotide reagents, there are also known phosphoramidite compounds of the following formula (I-1) where R2xe2x80x2 and R3xe2x80x2 are both hydrogen atom and Y is a dialkylamino group. See H. Koester et al, xe2x80x9cTetrahedron Lett.xe2x80x9d, 52, 5843 (1983) supra and PCT WO97/42202 supra. 
When the above phosphoramidite compounds are used as intermediates for DNA oligomers, generally, a 2-cyanoethoxydialkylaminophosphine derivative is reacted with a 5xe2x80x2-O- and base-protected nucleoside to make a DNA synthesis reagent.
According to this method, starting materials for chemical synthesis of DNA can be stably obtained, but in order to obtain the desired products in a high purity, by-products and impurities must be removed and this causes complexity in operation and increase of cost.
Therefore, in situ DNA synthesis reagents are needed, which do not require any steps of isolation and purification in preparation, and can be used as they are in a form of reacted solution for further reaction of synthesis of DNA oligomers.
Furthermore, the above conventional method has severe restrictions. One of them is that since accurate control of reaction on the solid phase is very difficult, particularly when the method is designed on a desired reaction scale, it is very difficult to set conditions therefor. Moreover, the porous glass is very expensive. Furthermore, since it is fundamental to use the reagents in excess amounts, this method is economically very disadvantageous when oligodeoxyribonucleotides are to be obtained in large amounts.
Specifically, it is technically not easy and requires much cost to design and practice the reaction in such a scale as exceeding 1 mmol utilizing a porous glass. These problems mean that if oligodeoxyribonucleotides were utilized for the pharmaceutical use, supply of them would be actually difficult.
In the production of oligodeoxyribonucleotides and oligoribonucleotides, there are demanded novel nucleoside compounds which can be utilized as starting materials for obtaining oligonucleotide in large amounts or as building blocks for extension of chains by use of easily available solid phase carriers, thereby facilitating separation and purification of intermediate products.
Furthermore, all of the steps in the above-mentioned conventional synthesis method are constructed of consecutive reactions and, hence, oligonucleotides having the desired sequences cannot be obtained unless all of the steps proceed with reaction yields of 100% or extremely close to 100%. Especially, the phosphorylation reaction step (condensation reaction) which is a step of extension of nucleotide chains gives a yield of 98.5-99.5 for each extension reation even in the present highest level, and this reaction yield determines the total yield of oligonucleotides having a desired sequence.
Recently, there is a report that a synthesis method in which a dimer nucleotide is used as a building block in building nucleotide chains thereby reducing the number of condensation is effective for improving the total yield (Krots et al, xe2x80x9cBioorg. Med. Chem. Lett.xe2x80x9d, 1997, 7, 73-78). Furthermore, there are many chemical synthesis methods for oligonucleotides using dimers or higher as building blocks (for example, xe2x80x9cChemistry of Nucleic Acid and Molecular Biology-Elements of Chemistry 46xe2x80x9d edited by Japan Chemical Association, and published by Gakkai Shuppan Center, 1985, pp.209-240). However, in the case of these building blocks, a series of the steps of protection-deprotection and phosphorylation are very complex and furthermore the operations such as extraction and chromatography are necessary in each step for the removal of by-products and impurities. These cause not only complexity of operation, but also increase the cost for the synthesis of building blocks.
Hitherto, the compound (Vxe2x80x2) in the following formula (X) has been known as a nucleotide block which is an intermediate starting material for DNA oligomers, and as a method for synthesizing this compound, there is known a method which comprises the steps of reacting phosphoramidite compound (Ixe2x80x2) with a nucleoside derivative (IX), oxidizing the nucleotide bond of the resulting nucleotide, and removing the protective group for 3xe2x80x2-position hydroxyl group from the resulting nucleotide derivative as shown in the following formula (X) (Refer to Japanese Patent Kohyo No.08-507752 corresponding to PCT/WO94/15946). 
In the above formula (X), B1 and B3 are bases protected with protective groups common in nucleotide chemistry, A1 and A3 each represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group, R1 and R4 each represents a protective group common in nucleotide chemistry, and X represents a dialkylamino group.
According to the above method, the nucleoside derivative represented by the formula (IX) which is a starting material must be produced in accordance with the reaction in the following formula (XI), and, in addition, the protective group R4 must be removed from the nucleotide block derivative after dimerization reaction of nucleotide.
Therefore, in order to obtain a desired nucleotide block at high purity, the synthesis steps need many stages, and furthermore by-products or impurities must be removed thereby causing complexity of operation and increase of the cost. 
In the above formula (XI), B3, R1, R4 and A3 are the same as defined in the formula (X).
Therefore, there are demanded a novel nucleotide compound and nucleotide block from which a nucleotide block useful for preparation of DNA oligomers can be simply produced without complex synthesis step and isolation and purification steps, as well as a method for producing an oligonucleotide using the same.
The object of the present invention is to provide nucleotide compounds and nucleotide blocks which meet the above-mentioned various demand, and a method for producing the same, and a method for producing an oligonucleotide using the nucleotide block.
As a result of intensive research conducted by the inventors in an attempt to solve the above problems, it has been found that when a nucleotide derivative represented by the following formula (I) is used, the above problems can be solved. Thus, the present invention has been accomplished.
That is, the present invention is a nucleotide compound represented by the following formula (I). 
In the above formula (I), R1 represents a protective group or an organic group represented by the formula xe2x80x94C(xe2x95x90O)-Axe2x80x2xe2x80x94(OCH2CH2)kOCH3 (in which k represents an integer of 3 or more, and Axe2x80x2 is a divalent organic group; R2, R2xe2x80x2, R3 and R3xe2x80x2 each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom and R2, R2xe2x80x2, R3 and R3xe2x80x2 may be the same or different; B1 and B2 each represents a base, if necessary, protected by a protective group common in nucleotide chemistry or Bxe2x80x2 (Bxe2x80x2xe2x95x90B1xe2x80x2xe2x80x94C(xe2x95x90O)-A-(OCH2CH2)kOCH3 in which B1xe2x80x2 represents one of the groups represented by the following formula (1), k represents an. integer of 3 or more, A is a divalent group and represents an arylene group or an alkylene group having a straight or branched chain which may contain a hetero-atom); X represents an oxygen atom or a sulfur atom; Y represents an azolyl group, a monoalkylamino group represented by HNR5 (in which R5 is an alkyl group or a cycloalkyl group), a dialkylamino group or a saturated nitrogenous heterocyclic ring; A1 and 2 each represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and n represents 0 or an integer of 1 to 100, with a proviso that the cases of the combinations (R2 and R3) and (R2xe2x80x2 and R3) being (hydrogen atom and hydrogen atom), (hydrogen atom and methyl group), (hydrogen atom and ethyl group), (methyl group and methyl group), (methyl group and ethyl group) or (ethyl group and ethyl group) are excluded. 
The inventors have further found that the above problems can be solved when the nucleotide compound represented by the above formula (I) is reacted with a 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside derivative or nucleotide derivative represented by the following formula (II) to synthesize a novel nucleotide block or oligonucleotide having the structure represented by the following formula (IV) through a novel nucleotide having the structure represented by the following formula (III). Thus, the present invention has been accomplished.
That is, the present invention is a method for producing a nucleotide block or oligonucleotide represented by the following formula (IV), characterized in that a nucleotide compound represented by the following formula (I) is reacted with a 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside derivative or nucleotide derivative represented by the following formula (II) and a trivalent phosphorus atom of the resulting nucleotide represented by the following formula (III) is oxidized or sulfurized to pentavalent phosphorus atom. 
In the above formulas (I-4), (II), (III) and (IV), R1 represents a protective group or an organic group represented by the formula xe2x80x94C(xe2x95x90O)-Axe2x80x2-(OCH2CH2)kOCH3 (in which k represents an integer of 3 or more, and Axe2x80x2 is a divalent organic group); R2, R2xe2x80x2, R2xe2x80x3, R3, R3xe2x80x3 and R3xe2x80x2 each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom and R2, R2xe2x80x2, R2xe2x80x3, R3, R3xe2x80x2 and R3xe2x80x3 may be the same or different; B1, B2, B3 and B4each represents a base, if necessary, protected by a protective group common in nucleotide chemistry or Bxe2x80x2 (Bxe2x80x2xe2x95x90B1xe2x80x2xe2x80x94C (xe2x95x90O)-A-(OCH2CH2)kOCH3 in which B1xe2x80x2 represents one of the groups represented by the following formula (1), k represents an integer of 3 or more, A is a divalent group and represents an arylene group or an alkylene group having a straight or branched chain which may contain a hetero-atom); X, Xxe2x80x2 and Xxe2x80x3 each represents an oxygen atom or a sulfur atom; Y represents an azolyl group; A1, A21, A3 and A4 each represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and m and n each represents 0 or an integer of 1 to 100, with a proviso that the cases of the combinations (R2 and R3), (R2xe2x80x2 and R3xe2x80x2) and (R2xe2x80x3 and R3xe2x80x3) being (hydrogen atom and hydrogen atom), (hydrogen atom and methyl group), (hydrogen atom and ethyl group), (methyl group and methyl group), (methyl group and ethyl group) or (ethyl group and ethyl group) are excluded. 
Furthermore, in the production method of the present invention, the compounds represented by the above formulas (III) and (IV) are novel nucleotide compounds.
(1) Among the nucleotide compounds represented by the above formula (I), the phosphorazolide compounds represented by the following formula (I-1) are suitable as in situ DNA synthesis reagents which can be used for further synthesis of DNA oligomers after synthesized without any isolation and purification step. 
(wherein B1 represents a base, if necessary, protected by a protective group common in nucleotide chemistry; R2xe2x80x2 and R3xe2x80x2 each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom; R1 represents a protective group or an organic group defined in the above formula (I); A1 represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and Y represents an azolyl group; with a proviso that the cases of the combinations (R2xe2x80x2 and R3xe2x80x2) being (hydrogen atom and hydrogen atom), (hydrogen atom and methyl group), (hydrogen atom and ethyl group), (methyl group and methyl group), (methyl group and ethyl group) or (ethyl group and ethyl group) are excluded.)
In the compounds represented by the above formula (I-1), the bases represented by B1 are known ones and as examples of the bases, mention may be made of purine derivatives such as derivatives of adenine, guanine and hypoxanthine and pyrimidine derivatives such as derivatives of cytosine, thymine and uracil. Specific examples are 1-thyminyl group, 1-(N-4-benzoylcytosinyl) group, 9-(N-6-benzoyladeninyl) group and 9-(N-2-isobutyrylguaninyl) group.
As R2xe2x80x2 and R3xe2x80x2 in the formula (I-1), mention may be made of hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, 1-methylbutyl group, 1-ethylpropyl group, cyclohexyl group, n-nonyl group, 2-phenylethyl group, 2-(methylthio)ethyl group, phenyl group, 1,1-diethyl-3-butenyl group and 1,1-dimethyl-2-phenylethyl group, excluding the cases of the combinations (R2 and R3xe2x80x2) being (hydrogen atom and hydrogen atom), (hydrogen atom and methyl group), (hydrogen atom and ethyl group), (methyl group and methyl group), (methyl group and ethyl group) or (ethyl group and ethyl group).
R1 includes, for example, trityl group, 4-methoxytrityl group, and 4,4xe2x80x2-dimethoxytrityl group, and, besides, succinyl group having polyethylene glycol methyl ether residue at one end, and Axe2x80x2 includes, for example, 1,4-phenylene group, methylene group and 1,1-dimethylethylene group, andY includes, for example, imidazolyl group, 2-methylimidazolyl group, 4-methylimidazolyl group, and 2,4-dimethylimidazolyl group.
A1 is hydrogen atom, hydroxyl group, alkoxy group and trialkylsilyloxy group, and alkoxy group includes, for example, methoxy group and ethoxy group, and trialkylsilyloxy group includes, for example, tert-butyldimethylsilyloxy group.
The phosphorazolide compound in the present invention can be easily produced by the reaction of a 5xe2x80x2-O- and base-protected nucleoside represented by the following formula (1-3) with an organooxybisazolylphosphine represented by the following formula (1-4) See the following formula (1-5). 
(wherein A1, B1 and R1 are the same as defined in the above formula (I-1). 
(wherein R2xe2x80x2, R3xe2x80x2 and Y are the same as defined in the above formula (I-1)). 
(wherein A1, B1, R2xe2x80x2, R3xe2x80x2, R1 and Y are the same as defined in the above formula (I-1)).
The above reaction is carried out in the following manner. 5xe2x80x2-O- and base-protected nucleoside is vacuum dried or is dissolved in an organic solvent such as pyridine or 1,4-dioxane, followed subjecting to azeotropic dehydration, and then it is mixed with an organooxybisazolylphosphine in an amount of 0.9-1.2 equivalent to the 5xe2x80x2-O- and base-protected nucleoside in an organic solvent such as toluene, pyridine, tetrahydrofuran, chloroform or acetonitrile under the condition of xe2x88x9280xc2x0 C. to room temperature. The reaction at lower temperature gives a higher yield of the phosphorazolide compound. Preferably, the organic solvent is one which has previously been dried with a drying agent and purified by distillation.
The completion of the reaction can be confirmed by measuring 31P-NMR spectrum of the reaction mixture. This reaction mixture can be used as it is as an in situ DNA synthesis reagent for synthesis of oligonucleotide.
On the other hand, the organooxybisazolylphosphine represented by the above formula (1-4) can be easily produced by the reaction of an organooxydichlorophosphine represented by the following formula (1-6) with an N-trimethylsilylazole compound represented by the following formula (1-7). See the following formula (1-8). 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula (I-1)).
xe2x80x83(CH3)3SiYxe2x80x83xe2x80x83(1-7)
(wherein Y is the same as defined in the above formula (I-1)). 
(wherein R2xe2x80x2, R3xe2x80x2 and Y are the same as defined in the above formula (I-1)).
The reaction represented by the above formula (1-8) is carried out by mixing an organooxydichlorophosphine with N-trimethylsilylazole compound in an amount of 2-3 equivalents to the organooxydichlorophosphine in toluene or a halogen organic solvent such as chloroform under the condition of room temperature. The reaction mixture is subjected to measurement of 1H-NMR spectrum to confirm completion of the reaction, and thereafter, the by-product chlorotrimethylsilane, the reaction solvent, the excess N-trimethylsilylazole compound and others are removed under reduced pressure to obtain the desired organooxybisazolylphosphine. Preferably the organic solvent is one which has previously been dried with a drying agent and purified by distillation.
The organooxydichlorophosphine represented by the above formula (1-6) can be easily produced by the reaction of the organooxytrimethylsilane represented by the following formula (1-9) with phosphorus trichloride. See the following formula (1-10) and Tsujiaki Hata et al, xe2x80x9cNucleic Acids Res.xe2x80x9d, 17, 8581 (1989) 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula (I-1)). 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula AE (I-1)).
The above reaction can be performed, for example, by mixing the above organooxytrimethylsilane with phosphorus trichloride in an amount of 2-5 equivalents to the organooxytrimethylsilane under the condition of 0xc2x0 C., and leaving the product at room temperature for 1 hour to 10 days. The resulting product is subjected to conventional distillation under reduced pressure to obtain the above organooxydichlorophosphine.
The above organooxytrimethylsilane represented by the above formula (1-9) can be easily produced by the reaction of a 2-cyanoethanol derivative represented by the following formula (1-11) with 1,1,1,3,3,3-hexamethyldisilazane (the following formula (1-12)) or by the reaction of chlorotrimethylsilane with a reaction product of an aldehyde or ketone represented by the following formula (1-13) with a cyanomethyl alkali metal compound such as cyanomethyl lithium (the following formula (1-14)). The cyanomethyl alkali metal compound can be easily produced by alkali metallization of active hydrogen adjacent to cyano group of acetonitrile with n-butyl lithium or the like. See the following formula (1-15). 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula (I-1)) . 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula (I-1)).
The reaction represented by the above formula (1-12) can be performed by mixing a 2-cyanoethanol derivative with 1,1,1,3,3,3-hexamethyldisilazane in an amount of 1-2 equivalents to the 2-cyanoethanol derivative and imidazole in an amount of 0.005-0.1 equivalent to the 2-cyanoethanol derivative, followed by refluxing under heating with stirring for 1-5 hours. The resulting product is subjected to conventional distillation under reduced pressure to obtain the desired above organooxytrimethylsilane. 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula (I-1)) 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula (I-1))
CH3CN+CH3CH2CH2CH2Lixe2x86x92LICH2CN+CH3CH2CH2CH3xe2x80x83xe2x80x83(1-15)
The reactions of the above formulas (1-14) and (1-15) are carried out in the following manner. First, to a solution of n-butyl lithium in n-hexane/tetrahydrofuran (ratio being 1/2) is added acetonitrile in an amount of 1.0-1.2 equivalents at the condition of xe2x88x9280xc2x0 C. to xe2x88x9260xc2x0 C., followed by stirring for 0.5-2 hours to carry out the reaction to obtain a solution of cyanomethyl lithium in n-hexane/tetrahydrofuran. Thereto is added the above aldehyde or ketone in an amount of 1.0-1.2 equivalents at the condition of xe2x88x9280xc2x0 C. to xe2x88x9260xc2x0 C., and the reaction temperature is returned to room temperature over a period of 0.5-1 hour, followed by stirring with addition of 1.2-1.5 equivalents of chlorotrimethylsilane. Preferably the organic solvent is one which has previously been dried with a drying agent and purified by distillation. Furthermore, the resulting product is subjected to conventional distillation under reduced pressure to obtain the desired organooxytrimethylsilane.
The N-trimethylsilylazole compound represented by the above formula (1-7) can be easily produced by the reaction of an azole represented by the following formula (1-16) with 1,1,1,3,3,3-hexamethyldisilazane. See the following formula (1-17).
Hyxe2x80x83xe2x80x83(1-16)
(wherein Y is the same as defined in the above formula (I-1)).
2HY+[(CH3)3)Si]2NHxe2x86x922(CH3)3SIY+NH3xe2x80x83xe2x80x83(1-17)
(wherein Y is the same as defined in the above formula (I-1)).
The above reaction can be performed by mixing an azole represented by the formula (1-16) with 1,1,1,3,3,3-hexamethyldisilazane in an amount of 1-2 equivalents to the azole, followed by refluxing under heating with stirring for 3-24 hours. The resulting product is subjected to conventional distillation under reduced pressure to obtain the N-trimethylsilylazole compound.
The phosphorazolide compound represented by the formula (I-1) is useful as an intermediate starting material in chemical synthesis of oligonucleotide, and, for example, DNA oligomers can be obtained in a high yield by using a reaction mixture of 3xe2x80x2-O-4-methylimidazolylphosphine derivative obtained by reacting 2-cyano-1-(1,1-diethyl-3-butenyl)ethoxybis(4-methylimidazolyl)phosphine with 5xe2x80x2-O-(4,4xe2x80x2-dimethoxytrityl) thymidine, for solid phase synthesis on a DNA automatic synthesizer in situ.
Yield of this reaction has a correlation with sum of van der Waals volumes of R2xe2x80x2 and R3xe2x80x2 of the above formula (I-1), and for the reason of high reaction yields, compounds of 49(angstrom)3 or larger in the sum of van der Waals volumes of R2xe2x80x2 and R3xe2x80x2 of the above formula (I-1) calculated under the following conditions are preferred. Examples of such compounds are those of R2xe2x80x2 being hydrogen atom and R3xe2x80x2 being n-propyl group.
Method for Calculation of the Van der Waals Volumes
In organooxybis(4-methylimidazolyl)phosphine represented by the following formula (2), first, three-dimensional molecular structure is determined by SPARTAN(trademark) Version 4.1.1 (Wavefunction, Inc.) and steric energy is optimized using MM force field, and, thereafter, steric structure is established by semiempirical molecular orbital method (AM1) . Then, the van der Waals volumes of R2xe2x80x2 and R3xe2x80x2 are obtained by the molecular volume calculation program of TSAR(trademark)3.0 (Oxford Molecular Group) on the basis of the steric structure obtained by the AM1. 
(wherein R2xe2x80x2 and R3xe2x80x2 are the same as defined in the above formula (I-1)).
(2) Like the compounds represented by the above formula (I-1), among the nucleotide compounds represented by the above formula (I), the monoalkylamino and dialkylamino type phosphoramidite compounds represented by the following formula (I-2) are suitable as in situ DNA synthesis reagents which can be used as they are in the reacted state for further synthesis of DNA oligomers without any isolation and purification step. 
(wherein B1 represents a base, if necessary, protected by a protective group common in nucleotide chemistry; R2xe2x80x2 and R3xe2x80x2 each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom; R1 represents a protective group or an organic group defined in the above formula (I); A1 represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and Y represents a monoalkylamino group represented by HNR5 (R5 represents an alkyl group or a cycloalkyl group) or dialkylamino group; with a proviso that the cases of the combinations (R2xe2x80x2 and R3xe2x80x2) being (hydrogen atom and hydrogen atom), (hydrogen atom and methyl group), (hydrogen atom and ethyl group), (methyl group and methyl group), (methyl group and ethyl group) or (ethyl group and ethyl group) are excluded.)
In the compounds represented by the formula (I-2), as examples of A1xe2x80x2, B1xe2x80x2, R2xe2x80x2, R3xe2x80x2 and R1xe2x80x2, mention may be made of those of the formula (I-1), and as examples of Y, mention may be made of isopropylamino group, n-butylamino group, isobutylamino group, tert-butylamino group, neopentylamino group, cyclohexylamino group, dimethylamino group and diisopropylamino group.
The monoalkylamino type and dialkylamino type phosphoramidite compounds as represented by the above formula (I-2)can be easily produced by the reaction of nucleotide derivatives represented by the following formula (I-1xe2x80x2) with monoalkylamines or dialkylamine represented by the following formula (2-4). Seethe following formula (2-5). 
(wherein A1, B1, R2xe2x80x2, R3xe2x80x2 and R1xe2x80x2 are the same as defined in the above formula (I-2), and Yxe2x80x2 represents an azolyl group such as imidazolyl group, 2-methylimidazolyl group or 4-methylimidazolyl group).
HYxe2x80x83xe2x80x83(2-4)
(wherein Y is the same as defined in the above formula (I-2)). 
(wherein A1, B1, R2xe2x80x2, R3xe2x80x2, R1 and Y are the same as defined in the above formula (I-2), and Yxe2x80x2 is the same as defined in the above formula (I-1xe2x80x2)).
The nucleotide derivatives represented by the above formula (I-1xe2x80x2) can be produced by the same method as for the compounds represented by the above formula (I-1). See the above formula (1-5).
The reaction represented by the above formula (2-5) is performed by adding to a reaction mixture resulting from the above mentioned preparation of the compound (I-1xe2x80x2) a monoalkylamine or dialkylamine in an amount of 0.9-1.2 equivalent to the compound (I-1xe2x80x2). The resulting monoalkylamino type and dialkylamine type phosphoramidite compounds in the form of a reaction mixture can be used as it is without isolation and purification as an in situ DNA synthesis reagent for synthesis of oligonucleotide.
The monoalkylamino type and dialkylamino type phosphoramidite compound represented by the formula (I-2) is useful as an intermediate starting material in chemical synthesis of oligonucleotide, and, for example, a reaction of 2-cyano-1-tert-butylethoxybis(4-methylimidazolyl)phosphine with 5xe2x80x2-O-(4,4xe2x80x2-dimethoxytrityl)thymidine provides 3xe2x80x2-O-4-methylimidazolylphosphine derivative of the nucleoside in a high yield. When isopropylamine is added to the above obtained reaction mixture, 3xe2x80x2-O-isopropylamino type phosphoramidite derivative of the nucleoside can be quantitatively obtained. Furthermore, when the reaction mixture is used as it is in situ for solid phase synthesis on a DNA automatic synthesizer using tetrazole as a catalyst, DNA oligomers can be obtained in a high yield.
Among the monoalkylamino type and dialkylamino type phosphoramidite compounds of the formula (I-2), from the point of the yield of DNA oligomers, preferred-are those of 49(angstrom)3 or larger in the sum of van der Waals volumes of the substituents R2xe2x80x2 and R3xe2x80x2 of the formula (I-2). Examples are monoalkylamino type phosphoramidite compounds of R2xe2x80x2 being hydrogen atom and R3xe2x80x2 being n-propyl group. The van der Waals volumes are calculated by the calculation method mentioned above concerning the-compound of the formula (I-1).
(3) Among the nucleotide compounds represented by the above formula (I), nucleotide compounds as represented by the following formula (I-3) in which polyethylene glycol (PEG) is introduced into the protective group of nucleic acid base can be separated and purified utilizing the properties of polyethylene glycol during the production of DNA synthesis reagents and are advantageous in that DNA oligomer products obtained therefrom are easy to handle as compared with those produced by conventional methods. 
(wherein B1xe2x80x2 represents a nucleic acid base commonly used in nucleotide chemistry; R2xe2x80x2 and R3xe2x80x2 each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom and R2xe2x80x2 and R3xe2x80x2 may be the same or different; R1 represents a protective group or an organic group defined in the above formula (I); A1 represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and Y represents a monoalkylamino group, a dialkylamino group, an azolyl group or a saturated nitrogenous heterocyclic ring; k represents an integer of 3 or more; and A is a divalent group and represents an arylene group or an alkylene group having straight or branched chain which may contain a hetero-atom).
As can be seen from the above formula (I-3), the nucleotide compounds are compounds containing a polyethylene glycol chain as the protective group for amino group or imino group of nucleic acid base.
As B1xe2x80x2 in the above formula (I-3), mention may be made of derivatives of adenine, guanine, cytosine, thymine and uracil represented by the following formula (1). 
Examples of R1, R2xe2x80x2 and R3xe2x80x2 and A1 in the above formula (I-3) are the same as those in the above formula (I-1), examples of Y are the same as those in the above formulas (I-1) and (I-2), and examples of A and Axe2x80x2 are 1,4-phenylene group, methylene group and dimethylethylene group.
Furthermore, the nucleoside compounds represented by the below-mentioned formula (I-3xe2x80x2) which are precursors of the above formula (I-3) or synthesis intermediates are also novel compounds.
The nucleotide compounds represented by the formula (I-3) are obtained by reacting nucleosides represented by the following formula (3-3) which are those of R1 being a protective group and R4 being hydrogen atom in the below-mentioned formula (I-3xe2x80x2) with a suitable phosphorylating agent. 
(wherein B1xe2x80x2, R1, A1, A and k are the same as defined in the above formula (I-3)).
For the reaction with the phosphorylating agent, the reaction formula (1-5) shown before can be used, and, furthermore, in the case of the compounds where Y is a monoalkyl group or a dialkyl group, the reaction formula (2-5) can be used.
The nucleotide compound of the formula (I-3) obtained by the above reaction may be used for the subsequent reaction as it is without separation and purification. For example, under the condition of xe2x88x9280xc2x0 C. to room temperature, the reaction mixture is mixed and reacted with a nucleoside compound at least only the base of which is suitably protected and which is subjected to vacuum drying or azeotropic dehydration and is in the form of a solution in an organic solvent such as toluene, pyridine, tetrahydrofuran, chloroform or acetonitrile, whereby dimerization reaction is performed. In this case, the 3xe2x80x2 hydroxyl group of the nucleoside used in the above reaction is not necessarily protected.
The nucleotide compound of the formula (I-3) is soluble in acetonitrile, tetrahydrofuran, pyridine and organic chlorine solvents, and can be reacted with the 3xe2x80x2-O- and 5xe2x80x2-O-unprotected second nucleoside in an above mentioned solvent having good affinity with polyethylene glycol, whereby a dimer can be obtained. On the other hand, when to the solution containing the dimer component is added a nonsolvent for polyethylene glycol, such as diethyl ether or diisopropyl ether in an amount of 5-20 times the volume of the solution, the dimer component is precipitated and can be easily recovered. Therefore, most of the separation-purification operations which have been needed for obtaining a dimer component from a conventional protected nucleotide with no polyethylene glycol moiety are eliminated. Besides, the present separation-purification method is sufficient for the subsequent building of nucleotide chains as well as for obtaining nucleotide dimer on a large scale. Accordingly, the present invention makes it possible to provide a very simple method for synthesis of building blocks of nucleotide dimers or oligomers and oligonucleotides.
(4) Moreover, the compounds of the formula (3-3) which are precursors for the compounds of the formula (I-3) can be easily synthesized through the compounds represented by the following formula (I-3xe2x80x2). 
(wherein R1 and R4 each represents a hydrogen atom or a protective group commonly used in nucleotide chemistry; A1 represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; A is a divalent group and represents an arylene group or an alkylene group having a straight or branched chain which may contain a hetero-atom; B1xe2x80x2 represents one of the groups represented by the following formula (1); and k represents an integer of 3 or more). 
As can be seen from the above formula (I-3xe2x80x2), the nucleoside compound is a compound characterized by containing a polyethylene glycol chain at the protective group for amino group or imino group of nucleic acid base.
As B1xe2x80x2 in the above formula (I-3xe2x80x2), mention may be made of derivatives of adenine, guanine, cytosine, thymine and uracil represented by the above formula (1). The protective groups commonly used in nucleotide chemistry for R1 and R4 include, for example, 4,4xe2x80x2-dimethoxytrityl group, trimethylsilyl group and t-butyldimethylsilyl group, and A includes, for example, 1,4-phenylene group, methylene group and dimethylethylene group.
The nucleoside compound in the above formula (I-3xe2x80x2) can be produced, for example, from a nucleoside represented by the following formula (4-3) and a carboxylic acid derivative of polyethylene glycol represented by the following formula (4-4-4). 
(wherein B1xe2x80x2 and A1 are the same as defined in the above formula (I-3xe2x80x2)).
CH3O(CH2CH2O)KHxe2x80x83xe2x80x83(4-4-1)
xe2x86x92CH3O(CH2CH2O)KSO2C6H4CH3xe2x80x83xe2x80x83(4-4-2)
xe2x86x92CH3O(CH2CH2O)Kxe2x86x92Axe2x86x92COOHxe2x80x83xe2x80x83(4-4-3)
xe2x86x92CH3O(CH2CH2O)Kxe2x86x92Axe2x86x92COZxe2x80x83xe2x80x83(4-4-4)
(wherein Z represents a halogen atom, an acyloxy group or an azolyl group, and A and k are the same as defined in the above formula (I-3xe2x80x2)).
Furthermore, the nucleoside represented by the above formula (4-3) in which the hydroxyl groups are protected with trimethylsilyl groups can be produced by the following method. That is, nucleoside subjected to azeotropy with pyridine is suspended in pyridine in an inert gas atmosphere, followed by reacting with chlorotrimethylsilane in an amount of 3-5 equivalents under the condition of 0xc2x0 C. to room temperature for 15-30 minutes. The reaction mixture can be used for the subsequent reaction without purification. See G. S. Ti et al, xe2x80x9cJ. Amer. Chem. Soc.xe2x80x9d, 104, 1316 (1982).
Moreover, the carboxylic acid derivative of polyethylene glycol which is represented by the above formula (4-4-4) can be produced by the following method. First, commercially available polyethylene glycol monomethyl ether (4-4-1) is reacted with tosyl chloride in the presence of sodium hydroxide to obtain a tosylated product (4-4-2) of polyethylene glycol monomethyl ether.
Then, in the presence of a base such as potassium carbonate, potassium-tert-butoxide and sodium hydride, the above tosylated product and an ester having an alcohol or a phenolic hydroxyl group such as methyl 4-hydroxybenzoate and methyl glucolate are refluxed under heating in acetonitrile or tetrahydrofuran to convert the tosylated product to an etherified product. The etherified product is subjected to alkali hydrolysis to obtain polyethylene glycol monomethyl ether having a carboxyl group at the end (4-4-3).
The resulting polyethylene glycol derivative is subjected to heat treatment with thionyl chloride to obtain an acid chloride, or is reacted with pivaloyl chloride in the presence of a tertiary amine to obtain a mixed acid anhydride, or is reacted with N,Nxe2x80x2-carbodiimidazole to obtain an azole derivative (4-4-4).
The carboxylic acid derivative of polyethylene glycol which is represented by the above formula (4-4-4) is reacted with nucleoside having protected hydroxyl groups in an inert gas atmosphere at 0-40xc2x0 C. for about 2-24 hours, whereby the polyethylene glycol derivative can be introduced as a protective group for amino group or imino group of the nuclei acid base. When deprotection of the hydroxyl groups is effected, there is obtained 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside compound containing polyethylene glycol as a protective group for amino group or imino group of the nucleic acid base. When polyethylene glycol is introduced as a protective group for imino group, the presence of a tertiary amine such as diisopropylethylamine in this reaction results in rapid proceeding of the reaction. When 5xe2x80x2-hydroxyl group of the thus obtained 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside is selectively protected, the compound of the above formula (3-3) is easily obtained.
The production of the nucleoside compound obtained above can be confirmed by thin layer chromatography and the like. Its structure can be identified by measuring 1H-nuclear magnetic resonance (NMR) spectrum.
The nucleoside compound represented by the formula (I-3xe2x80x2) is a novel compound, and this compound is soluble in acetonitrile, tetrahydrofuran, pyridine, and organic chlorine solvents. When these solvents which have good affinity with polyethylene glycol are used, various reactions can completely be performed in a solution.
The nucleoside compound represented by the formula (I-3xe2x80x2) changes in its solubility depending on properties of a polyethylene glycol chain introduced into the site of the nuclei acid base. Addition of a suitable amount of, for example, diethyl ether or diisopropyl ether to the solution of the nucleoside compound or a derivative thereof, makes it possible to precipitate and recover the compound or derivative. It is also effective to carry out recrystallization from 2-propanol to recover the nucleoside compound or derivatives thereof. In this case, recovered product is of high purity.
The nucleoside compound represented by the formula (I-3xe2x80x2) can also be utilized as a starting material at a terminal site for synthesis of oligonucleotides or as a building block for building of nucleotide chain.
(5) Method for Producing Nucleotide Block and Oligonucleotide Using the Same
The method for producing nucleotide block and the method for producing oligonucleotide using the same according to the present invention is as follows. The nucleotide derivative represented by the above formula (I-4) is reacted with 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside derivative or nucleotide derivative represented by the above formula (II) optionally in the presence of a suitably selected activating agent to obtain a nucleotide having the structure represented by the above formula (III), and the compound represented by the formula (IV) is synthesized via said nucleotide of the formula (III). See the following formula (5-7).
Specifically, the compound (III) can be obtained by reacting the compounds (I-4) and (II) as they are, but the compound (III) can be further smoothly obtained by allowing an activating agent such as benzimidazole or benzotriazole to be present in the reaction of the compounds (I-4) and (II).
Further characteristic of this production method is that a phosphorus atom of the compound (III) obtained by the reaction of the compounds (I-4) and (II) or the reaction in the presence of a suitably selected activating agent is oxidized or sulfurized to prepare the compound (IV),and then this is isolated and reacted with a phosphorylating agent (5-III) as shown later in the formula (5-14), whereby again the (I-4) type compound can be obtained. This is further reacted with the (II) type compound, whereby the oligonucleotide chain can be sequentially extended.
In this case, when R1 of the compound (I-4) is a succinyl group having a polyethylene glycol methyl ether residue or at least one of the protective groups of bases has a polyethylene glycol methyl ether residue, the compound (III) resulting from the reaction of the compounds (I-4) and (II) can be recovered using a nonsolvent for polyethylene glycol, such as diethyl ether, then reacted with the phosphorylating agent (5-III), and further condensed with the compound (II). If this procedure is repeated, the (III) type compound of a desired chain length is obtained. If the trivalent phosphorus atom of the (III) type compound is converted to a pentavalent atom, the compound (IV) can also be obtained.
Moreover, in the above method for production of nucleotide block and oligonucleotide using the properties of the polyethylene glycol bearing protective group, 5xe2x80x2-hydroxyl group of the by-product that may result from the reaction with 3xe2x80x2-hydroxyl group of the compound (II) during the reaction of the compounds (I-4) and (II), may be masked with triethylsilyl group, triisopropylsilyl group or tert-butyldimethylsilyl group and the products may be recovered in a solvent such as diethyl ether, before the reaction with the phosphorylating agent (5-III) is effected. 
(in the above formula, B1, B2, B3, B4, R2, R3, R2xe2x80x2, R3xe2x80x2, R2xe2x80x2, R3xe2x80x2, R1, A1, A2, A3, A4, X, Xxe2x80x3 and n and m are the same as defined in the above formula (IV), and Y is the same as defined in the above formula (I-4)).
The method for the production of nucleotide blocks or oligonucleotides in the present invention is characterized in that a nucleotide derivative represented by the above formula (I-4) is reacted with a 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside derivative or nucleotide derivative represented by the above formula (II) to obtain the nucleotide represented by the above formula (III) and a trivalent phosphorus atom of the resulting nucleotide is oxidized or sulfurized to pentavalent phosphorus atom. The reaction formula is exemplified by the above formula (5-7). Furthermore, the present nucleotide block or oligonucleotide is a compound represented by the above formula (IV), and a precursor thereof is a nucleotide represented by the above formula (III).
As the bases represented by B1 and B2 in the above formula (I-4), mention may be made of purine derivatives such as derivatives of adenine, guanine and hypoxanthine, and pyrimidine derivatives such as derivatives of cytosine, thymine and uracil. Specific examples thereof are 1-thyminyl group, 1-(N-3-benzoylthyminyl) group, 1-(N-4-benzoylcytosinyl) group, 1-(N-4-anisoylcytosinyl) group, 9-(N-6-benzoyladeninyl) group, 9-(N-6, N-6-bisbenzoyladeninyl) group, and 9xe2x80x94(N-2-isobutyrylguaninyl) group, and Bxe2x80x2 represented by the following formula (4).
Bxe2x80x2=B1xe2x80x2-C(xe2x95x90O)-A-(OCH2CH2)kOCH3xe2x80x83xe2x80x83(4)
(in which B1xe2x80x2 represents one of the groups represented by the following formula (1), k represents an integer of 3 or more, A is a divalent group and represents an arylene group or an alkylene group having a straight or branched chain which may contain a hetero-atom). 
A and Axe2x80x2 each includes, for example, 1,4-phenylene group, methylene group and dimethylethylene group.
As the bases represented by B3 and B4 in the above formula (II), mention may be made of purine derivatives such as derivatives of adenine, guanine and hypoxanthine, and pyrimidine derivatives such as derivatives of cytosine, thymine and uracil. Specific examples thereof are 1-thyminyl group, 1-(N-3-benzoylthyminyl) group, 1-(N-4-benzoylcytosinyl) group, 1-(N-4-anisoylcytosinly) group, 9-(N-6-benzoyladeninyl) group, 9-(N-6, N-6-bisbenzoyladeninyl) group and 9-(N-2-isobutyrylguaninyl) group.
Furthermore, as R2, R3, R2xe2x80x2, R3xe2x80x2, R2xe2x80x3 and R3xe2x80x3 in the above formulas (I-4) and (II), mention may be made of, for example, hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, 1-ethylpropyl group, cyclohexyl group, n-nonyl group, 2-phenylethyl group, 2-(methylthio) ethyl group, phenyl group, 1,1-diethyl-3-butenyl group and/or 1,1-dimethyl-2-phenylethyl group. R1 in the above formula (I-4) includes, for example, trityl group, 4-methoxytrityl group, and 4,4xe2x80x2-dimethoxytrityl group besides, succinyl group having polyethylene glycol methyl ether residue at one end.
Y in the above formula (I-4) includes, for example, imidazolyl group, 2-methylimidazolyl group, 4-methylimidazolyl group, 2,4-dimethylimidazolyl group, triazolyl group, and other azolyl groups.
The alkoxy group represented by A1, A2, A3 and A4 in the above formulas (I-4) and (II) includes, for example, methoxy group and ethoxy group, and the trialkylsilyloxy group including, for example, tert-butyldimethylsilyloxy group.
The present nucleotide block and oligonucleotide can be easily produced by oxidizing or sulfurizing the trivalent phosphorus atom of the nucleotide represented by the formula (III) to a pentavalent atom. See the following formula (5-12). 
(in the above formula, B1, B2, B3, B4, R2, R3, R2xe2x80x2, R3xe2x80x2, R2xe2x80x3, R3xe2x80x3, R1, A1, A2, A3, A4, X, Xxe2x80x2, Xxe2x80x3 and n and m are the same as defined in the above formula (IV)).
Furthermore, the nucleotide represented by the above formula (I-4) can be easily produced in accordance with the reaction shown by the following formula (5-14). 
(in the above formula, B1, B2, R2, R3, R2xe2x80x2, R3xe2x80x2, R1 A1, A2, X and n are the same as defined in the above formula (IV), and Y is the same as defined in the above formula (I-4)).
The above reaction is attained in the following manner. That is, organooxydichlorophosphine represented by the formula (5-I) in the above formula (5-14) which is a starting material is reacted with N-trimethylsilylazole compound represented by the formula (5-II) to obtain organooxybisazolylphosphine represented by the formula (5-III). Then, 5xe2x80x2-O- and base-protected nucleoside derivative or 5xe2x80x2-O- and base-protected nucleotide derivative represented by the formula (5-IV) in the formula (5-14) is vacuum dried or is dissolved in an organic solvent such as pyridine or 1,4-dioxane and subjected to azeotropic dehydration, and then the product is mixed and reacted with the above organooxybisazolylphosphine in an amount of 0.8-1.2 equivalent to the 5xe2x80x2-O- and base-protected nucleoside or 5xe2x80x2-O- and base-protected nucleotide derivative in an organic solvent solution such as in toluene, pyridine, tetrahydrofuran, chloroform or acetonitrile under the condition of xe2x88x9280xc2x0 C. to room temperature. The reaction at lower temperature gives a higher yield of the nucleotide derivative represented by the above formula (I-4). Preferably, the organic solvent is one which has previously been dried with a drying agent and then purified by distillation. When chloroform is used as the organic solvent, the yield of the desired nucleotide is further improved. The completion of the reaction can be confirmed by measuring 31P-NMR spectrum of the reaction mixture. Since this reaction proceeds highly selectively from the starting organooxydichlorophosphine represented by the above formula (5-I) to the desired nucleotide, the synthesis can be performed in situ without isolation and purification, and the reaction mixture can be used in situ for the synthesis of nucleotide represented by the above formula (III).
The reaction of the above formula (5-7) can also be attained in the following manner. 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside derivative or nucleotide derivative represented by the formula (II) in an amount of 1-2 equivalents to the nucleotide represented by the above formula (I-4) is vacuum dried or is dissolved in an organic solvent such as pyridine and subjected to azeotropic dehydration, and then the product is mixed and reacted with the above nucleotide derivative in an organic solvent solution such as in pyridine, chloroform or acetonitrile under the condition of xe2x88x9280xc2x0 C. to room temperature, whereby the 5xe2x80x2-position hydroxyl group of the 3xe2x80x2-O- and 5xe2x80x2-O-unprotected-nucleoside derivative or nucleotide derivative is selectively reacted. Thus, the reaction is performed. Preferably, the organic solvent is one which has previously been dried with a drying agent and then purified by distillation. When pyridine is used as the organic solvent, the yield of the desired nucleotide represented by the above formula (III) is further improved. The completion of the reaction can be confirmed by measuring 31P-NMR spectrum of the reaction mixture. In this reaction, the higher the bulkiness of the substituents R2xe2x80x2 and R3xe2x80x2 in the formula (I-4) is, the higher the selectivity of coupling reaction with 5xe2x80x2 hydroxyl group is, and especially, when the sums of the van der Waals volume of R2xe2x80x2 and R3xe2x80x2 is 49 (angstrom)3 or more, the selectivity is excellent.
According to the method for producing the nucleotide block and the method for producing oligonucleotide using the same in the present invention, a nucleotide derivative represented by the above formula (I-4) is reacted with a 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleoside or nucleotide derivative to synthesize a compound represented by the above formula (IV) via a nucleotide having the structure represented by the formula (III), and especially when Y of the compound (I-4) is an imidazolyl group or a 4-methylimidazolyl group in the step of the reaction of the compound (I-4) with (II), the reaction can further smoothly produce the compound (III) if a reaction accelerator is allowed to be present.
Examples of the reaction accelerator are imidazole compounds such as benzimidazole and 3-nitroimidazole, and triazole compounds such as benzotriazole, 3-methyltriazole, and 3-nitrotriazole. Solvents used for the reaction are preferably chloroform, acetonitrile, toluene and pyridine. The reaction can be carried out at xe2x88x9280xc2x0 C. to 50xc2x0 C. Addition amount of the reaction accelerator is preferably 1-10 equivalents to the compound (I-4).
In the present invention, in the case of having a polyethylene glycol methyl ether residue as the protective group for 5xe2x80x2-hydroxyl group or base of the compound (I-4), the compound (III) resulting from the reaction of the compound (I-4) and the compound (II) can be recovered using a poor solvent for polyethylene glycol, such as diethyl ether, then reacted with phosphorylation agent (5-III), and further condensed with the compound (II). This procedure can be repeated to obtain a (III) type compound of a desired chain length, and finally the trivalent phosphorus atom is converted to a pentavalent atom to give the compound (IV).
In this process, 5xe2x80x2-hydroxyl group of by-products that may be produced by the reaction with 3xe2x80x2-hydroxyl group of the compound (II) during the reaction of the compound (I-4) with the compound (II), can be masked with triisopropylsilyl group or the like.
As the masking agent, chlorotriisopropylsilane, chloro-tert-butyldimethylsilane, chloro-tert-butyldiphenylsilane, and the like can be used, but for accelerating this reaction, there may be used together imidazole compounds such as imidazole, 2-methylimidazole and 2-ethylimidazole. And, silylation agents such as triethylsilylimidazole, tert-butyldimethylsilylimidazole and the like can be used as the masking agent.
The reaction solvents are preferably dimethylformamide, dichloromethane, chloroform, acetonitrile and the like. The reaction can be carried out at 0-50xc2x0 C. Amount of the solvent is preferably 0.1-0.5 equivalent to the compound (II).
The reaction of the above formula (5-12) can also be attained by mixing and reacting the nucleotide synthesized as above in the form of a reaction mixture without subjecting to isolation and purification with, for example, elemental sulfur in an amount of 1-3 equivalents to said nucleotide. The completion of the reaction can be confirmed by measuring 31P-NMR spectrum of the reaction mixture. The resulting nucleotide block can be readily isolated and purified by separation extraction and column chromatography at high purity.
Furthermore, the 3xe2x80x2-O- and 5xe2x80x2-O-unprotected nucleotide derivative represented by the above formula (II) which is another starting material in production of nucleotide can be easily produced by removing the protective group R1 from the nucleotide block or oligonucleotide represented by the above formula (IV).
The nucleotide and nucleotide block produced by the above method are useful as an intermediate for chemical synthesis of oligonucleotide.